Cloning of aquaporin-1 of the blue crab, Callinectes sapidus: its expression during the larval development in hyposalinity

Chung J, Maurer Leah, Bratcher Meagan, Pitula, Ogburn

Description

Ontogenetic variation in salinity adaptation has been noted for the blue crab, Callinectes sapidus , which uses the export strategy for larval development: females migrate from the estuaries to the coast to spawn, larvae develop in the ocean, and postlarvae (megalopae) colonize estuarine areas. We hypothesized that C. sapidus larvae may be stenohaline and have limited osmoregulatory capacity which compromises their ability to survive in lower salinity waters. We tested this hypothesis using hatchery-raised larvae that were traceable to specific life stages. In addition, we aimed to understand the possible involvement of AQP-1 in salinity adaptation during larval development and during exposure to hyposalinity. Results A full-length cDNA sequence of aquaporin (GenBank JQ970426) was isolated from the hypodermis of the blue crab, C. sapidus , using PCR with degenerate primers and 5′ and 3′ RACE. The open reading frame of CasAQP-1 consists of 238 amino acids containing six helical structures and two NPA motifs for the water pore. The expression pattern of CasAQP-1 was ubiquitous in cDNAs from all tissues examined, although higher in the hepatopancreas, thoracic ganglia, abdominal muscle, and hypodermis and lower in the antennal gland, heart, hemocytes, ovary, eyestalk, brain, hindgut, Y-organs, and gill. Callinectes larvae differed in their capacity to molt in hyposalinity, as those at earlier stages from Zoea (Z) 1 to Z4 had lower molting rates than those from Z5 onwards, as compared to controls kept in 30 ppt water. No difference was found in the survival of larvae held at 15 and 30 ppt. CasAQP-1 expression differed with ontogeny during larval development, with significantly higher expression at Z1-2, compared to other larval stages. The exposure to 15 ppt affected larval-stage dependent CasAQP-1 expression which was significantly higher in Z2- 6 stages than the other larval stages. Conclusions We report the ontogenetic variation in CasAQP-1 expression during the larval development of C. sapidus and the induction of its expression at early larval stages in the exposure of hyposalinity. However, it remains to be determined if the increase in CasAQP-1 expression at later larval stages may have a role in adaptation to hyposalinity.

Sujets

• Aquaporin
• Osmorégulation

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 298
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Chung et al. Aquatic Biosystems 2012, 8:21
http://www.aquaticbiosystems.org/content/8/1/21 AQUATIC BIOSYSTEMS
RESEARCH Open Access
Cloning of aquaporin-1 of the blue crab,
Callinectes sapidus: its expression during the larval
development in hyposalinity
4* 1 2 2 3J Sook Chung , Leah Maurer , Meagan Bratcher , Joseph S Pitula and Matthew B Ogburn
Abstract
Background: Ontogenetic variation in salinity adaptation has been noted for the blue crab, Callinectes sapidus,
which uses the export strategy for larval development: females migrate from the estuaries to the coast to spawn,
larvae develop in the ocean, and postlarvae (megalopae) colonize estuarine areas. We hypothesized that C. sapidus
larvae may be stenohaline and have limited osmoregulatory capacity which compromises their ability to survive in
lower salinity waters. We tested this hypothesis using hatchery-raised larvae that were traceable to specific life
stages. In addition, we aimed to understand the possible involvement of AQP-1 in salinity adaptation during larval
development and during exposure to hyposalinity.
Results: A full-length cDNA sequence of aquaporin (GenBank JQ970426) was isolated from the hypodermis of the
blue crab, C. sapidus, using PCR with degenerate primers and 50 and 30 RACE. The open reading frame of CasAQP-1
consists of 238 amino acids containing six helical structures and two NPA motifs for the water pore. The expression
pattern of CasAQP-1 was ubiquitous in cDNAs from all tissues examined, although higher in the hepatopancreas,
thoracic ganglia, abdominal muscle, and hypodermis and lower in the antennal gland, heart, hemocytes, ovary,
eyestalk, brain, hindgut, Y-organs, and gill. Callinectes larvae differed in their capacity to molt in hyposalinity, as
those at earlier stages from Zoea (Z) 1 to Z4 had lower molting rates than those from Z5 onwards, as compared to
controls kept in 30 ppt water. No difference was found in the survival of larvae held at 15 and 30 ppt. CasAQP-1
expression differed with ontogeny during larval development, with significantly higher expression at Z1-2,
compared to other larval stages. The exposure to 15 ppt affected larval-stage dependent CasAQP-1 expression
which was significantly higher in Z2- 6 stages than the other larval stages.
Conclusions: We report the ontogenetic variation in CasAQP-1 expression during the larval development of
C. sapidus and the induction of its expression at early larval stages in the exposure of hyposalinity. However, it
remains to be determined if the increase in CasAQP-1 expression at later larval stages may have a role in adaptation
to hyposalinity.
Keywords: Aquaporin, Blue crab larvae, Ontogenetic variation, Osmoregulation, Salinity tolerance
Background the American lobster, Homarus americanus [2,3] are
Ontogenetic variation in salinity tolerance and osmoregu- known to be strong hyper- and hypo-osmoregulators and
latorycapacitymaybedirectlyrelatedtopatternsofdisper- inhabit a wide range of salinities. On the other hand, their
sal and recruitment of animals in various aquatic habitats. embryonic and larval stages require high salinity water,
In decapod crustaceans, adults of the blue crab,Callinectes possibly due toa limited osmoregulatory capacity [4]. Con-
sapidus, the green shore crab, Carcinus maenas [1], and sequently, larvae are typically exported to higher salinity
waters for larval development either by migration of
females prior to spawning or rapid transport of larvae out* Correspondence: [e-mail]
4
Institute of Marine and Environmental Technology, University of Maryland of estuaries during ebb tides.
Center for Environmental Science, 701 East Pratt Street, Columbus Center,
In estuaries such as the Chesapeake Bay, life stage-
Suite 236, Baltimore, MD, USA
dependent osmoregulatory capacity and salinity toleranceFull list of author information is available at the end of the article
© 2012 Chung et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Chung et al. Aquatic Biosystems 2012, 8:21 Page 2 of 10
http://www.aquaticbiosystems.org/content/8/1/21
may be the driving force underlying population structures seawater (ASW) at 15 ppt showed significantly higher ex-
of C. sapidus, resulting from migration of adult females to pression of the blue crab aquaporin orthologue CasAQP-1
high salinity waters for spawning and the return migration as compared to those exposed to 30 ppt. Molting percent-
of postlarvae (megalopae). First, adult females migrate to age is much lower in ASW-exposed Z2-6 larvae asopposed
higher salinity areas near coastal waters after the pubertal- to those reared at 15 ppt, suggesting that energy reserves
terminal molting and mating, where they spawn and re- are diverted to survival through osmoregulation under
lease pelagic larvae [5]. These larvae largely spend seven- these conditions.
eight zoeal stages in coastal ocean waters [6]. However,
upon molting to the megalopa stage, they migrate back to Results
the coast and invade lower salinity estuarine areas where Sequence analyses of C. sapidus aquaporin 1 (CasAQP-1)
they metamorphose to the first crab stage [7]. Thus, the The nucleotide and deduced aa sequences of C. sapidus
life cycle of C. sapidus presents a typical ontogenetic vari- aquaporin-1 (CasAQP-1: GenBank JQ970426) are pre-
0 0
ationinsalinityadaptationas osmoregulatorycapacityand sented in Figure 1A. Both the 5 and 3 UTRs (italicized in
salinity tolerance are acquired during late larval develop- Figure 1A) contained three terminal oligopyrimidine tracts
mentor the megalopal stage. (TOP) as a translation regulatory site: two located in the
0
Salinityadaptationinvolvesacomplexprocessthatentails 50 UTR and one located in the 3 UTR (highlighted in bold
dramatic changes in cell volume, ion transport, cellular me- and underlined in Figure 1A). The deduced amino acid
tabolism, and whole-scale tissue remodeling. A large num- sequence of CasAQP-1 does not contain the signal pep-
ber of genes are involved in this osmoregulatory process in tide (P=0.068 by www.cbs.dtu.dk/services/SignalP). The
Carcinus maenas [8]. The aquaporin (AQP) family of water Conserved Domain database (www.ncbi.nlm.nih.gov/Struc-
channels, small and very hydrophobic intrinsic membrane ture/cdd/)identifiedfromthededucedaminoaregionfrom
proteins, is critical in the physiological processes of water amino acid E to V ofCasAQP-1asaputativemajorin-15 214
and solute transport for salinity adaptation [9]. AQPs are trinsic protein (MIP) superfamily member (Figure 1A,
ubiquitous, being present in bacteria, plants, and animals. marked with arrows). The two highly conserved hydropho-
To date, 13 isoforms of the AQP family can be grouped bic stretch regions, with two NPA boxes (boxed) that are
into three subfamilies: aquaporins, aquaglyceroporins, and involved in forming the water pore, are underlined. Four pu-
superaquaporins [10]. Among these three subfamilies, the tative phosphorylation sites were predicted by NetPhos 2.0
aquaporinsubfamilyincludingAQPs0,1,2,4,5,6,and8is Server (www.cbs.dtu.dk/services/NetPhos/) with a value >0.9
selective for water transport [11]. at three serine residues (S , ) and of 0.6 at one93, 199 and 224
The involvement of aquaporins in salinity adaptation has threonineresidue(T ).119
been most studied for AQP-1 in teleosts, although other The 3D structure of CasAQP-1 (Figure 1B) was obtained
aquaporins have been identified in this process. The ex- using the 3D structure of c1ymgA (PDB) as the template
pression of AQP-1 is found in most organs of fish with showing that 91% of 217 deduced aa of CasAQP-1 were
high expression in gill, intestine, and kidney, where the ex- modeled with 100% confidence by the single highest scoring
pression levels change in response to different salinities. template. 10 α helices including six transmembrane helices,
Acclimation to hyposalinity up-regulated AQP 1a expres- one β strand and 12 random coil secondary structures were
sion in the gill of Atlantic salmon and black porgy [12,13]. predicted. Color rainbows indicates N- to C-termini of
On the other hand, acclimation to hypersalinity increased CasAQP-1.
AQP-1 expression in the intestines and kidneys of Atlantic A phylogram was generated with the deduced aa
salmon [13], and in the intestines of European eels [14], sequences of 15 different AQP-1 including seven verte-
Japanese eels [15], and sea bass [16]. These studies demon- brates and 8 invertebrates (Figure 1C). The tree contains
strate the potential for the involvement of aquaporins in two separate clades: one for vertebrates and the other for
the adaptationof C. sapidustohyposalinity. invertebrates. CasAQP-1 was located close to AQP-1 of
In view of the fact that adult females migrate to high sal- the cockroach, Blattella germanica, both of which were
inity waters for spawning and that high salinity is required separate from the rest of the branch of insects and the
forlarvaldevelopment,wehypothesizedthat C. sapiduslar- water flea, Daphnia pulex.
vae may be stenohaline and have limited osmoregulatory
capacity which compromises their ability to survive in Spatial expression of C. sapidus aquaporin I (CasAQP-1)
lower salinity waters. We tested this hypothesis using The ex